The present invention is in the field of molecular biology; more particularly, the present invention describes the nucleic acid and amino acid sequences of three novel cellubrevin homologs.
Cellubrevins are homologs of synaptobrevins, synaptic vesicle-associated membrane proteins (VAMPs). Synaptobrevin was first discovered in rat brain (Baumert et al (1989) Embo J 8:379-84) and initially thought to be limited to neuronal cells. Synaptobrevin is an integral membrane protein of 18 kDA (Ralston E et al (1994) J Biol Chem 269:15403-6) involved in the movement of vesicles from the plasmalemma of one cell, across the synapse, to the plasma membrane of the receptive neuron. This regulated vesicle trafficking pathway and the endocytotic process may be blocked by the highly specific action of clostridial neurotoxins which prevents neurotransmitter release by cleaving the synaptobrevin molecule. Synaptobrevins are now known to occur and function in constitutive vesicle trafficking pathways involving the receptor-mediated endocytotic and exocytotic pathways of many non-neuronal cell types.
Cellubrevins are 16 kDa proteins first found and investigated in rat cells and tissues (McMahon HT et al (1993) Nature 364:346-9). In vitro studies of various cellular membranes (Galli et al (1994) J Cell Biol 125:1015-24; Link et al (1993) J Biol Chem 268:18423-6) have shown that VAMPS including the cellubrevins are widely distributed and are important in membrane trafficking. They appear to participate in axon extension via exocytosis during development, in the release of neurotransmitters and modulatory peptides, and in endocytosis. Endocytotic vesicular transport includes such intracellular events as the fusions and fissions of the nuclear membrane, endoplasmic reticulum, Golgi apparatus, and various inclusion bodies such as peroxisomes or lysosomes.
Endocytotic processes appear to be universal in eukaryotic cells as diverse as yeast, Caenorhabditis elegans, Drosophila, and humans. The homologous proteins which direct the movement of vesicles within and between the cells of these organisms contain evolutionarily conserved domains. Generally, VAMPs have a three domain organization. The domains include a variable proline-rich, N-terminal sequence of 28 amino acids, a highly conserved central hydrophilic core of 69 amino acids, and a hydrophobic sequence of 23 amino acids presumed to be the membrane anchor.
As mentioned for synaptobrevin above, cellubrevins are sensitive to selective proteolysis by metalloendoproteases such as the zinc endoprotease which comprises the light chain of tetanus toxin. Experiments have shown that endosome fusion may continue even after specific cellubrevin cleavage through temperature- and ATP-dependent docking and fusion processes involving N-ethylmaleimide-sensitive fusion proteins (NSF) and small, soluble attachment proteins (SNAP).
Because tissue distribution and VAMPs are more numerous and widely distributed than initially recognized, research on their differential expression and subcellular localization may turn out to be one of the most fruitful areas for the control or amelioration of diseases and disease symptoms.
Cellubrevins are associated with particular cell types, participate in both intracellular and extracellular pathways, and appear to vary in their abundance and specificity. Elucidation of the interactions of the novel cellubrevins (and associated VAMPs) with docking proteins such as syntaxin and SNAPs of the plasmalemma or the core fusion proteins such as NSF and the synaptotagmins (Bark I C and Wilson M C (1994) Proc Natl Acad Sci 91:4621-4624) provide means for the regulation of vesicle trafficking in normal as well as acute and chronic disease situations.
The subject invention provides nucleotide sequences which uniquely encode novel human cellubrevins. The cDNAs, disclosed herein, are designated: 1) cb-1 (SEQ ID NO:1) which was found within Incyte Clone No. 80184 and encodes a polypeptide designated CB-1 (SEQ ID NO:2); 2) cb-2 (SEQ ID NO:3) which was found within Incyte Clone No. 122826 and encodes a polypeptide designated CB-2 (SEQ ID NO:4), 3) cb-3 (SEQ ID NO:5) which was identified and extended using Incyte Clone No. 311537 and encodes a polypeptide designated CB-3 (SEQ ID NO:6); and 4) cb-4 (SEQ ID NO:7) which was found within Incyte Clone No. 674 719 and encodes a polypeptide designated CB-4 (SEQ ID NO:8).
The invention also comprises using these CBs or their variants to intercede in conditions involving physiologic or pathologic compromise which include the steps of testing a sample or an extract with cb nucleic acids, fragments or oligomers thereof. Aspects of the invention include the antisense of the nucleic acid sequences; cloning or expression vectors containing the nucleic acid sequences; host cells or organisms transformed with these expression vectors; a method for the production and recovery of purified CBs from host cells; and purified proteins which may be used to generate antibodies for diagnosis or therapy of activated or inflamed cells and/or tissues.